In 1975 Kohler and Milstein showed that somatic cell hybridizations could be used to establish continuous cell lines capable of secreting specific antibodies against predefined antigens. The antibodies produced by such cell lines are fundamentally different from polyclonal antisera obtained from conventionally immunized mammals. Each such cell line or hybridoma produces a homogeneous, or monoclonal, immunoglobulin that represents only one of the many antibodies produced by the immunized mammal. Thus, a perpetual supply of antibody of predetermined specificity may be created. Kohler, G. and Milstein, C. (1975) Nature 256:495-497.
The classic hybridoma technique is based on the fusion of mortal antibody-prooucing B lymphocytes from an immunized animal with immortal cells derived from a myeloma cell line. In usual practice, the myeloma cell line does not itself produce antibooies. When the non-producing variant (myeloma) for immunoglobulin production is fused with the antibody-producing cell such as a B lymphocyte, immunoglobulin production is not extinguished, nor is production (secretion) of immunoglobulin by the myeloma reactivated. Hybrids produced only those immunoglobulin chains that were being produced by the parental cells at the time of fusion.
The monoclonal antibodies produced by hybridomas have been widely used to elucidate the antigenic structure of viruses. These studies have focused attention on the potential of vaccination and the role of immunity in parasitic diseases. Yet study of protozoan parasites has been difficult because of the complex life cycle of these organisms. Since production of hybridomas does not require purified antigen, monoclonal antibodies offer a simple and attractive approach to the study of human parasitic disease.
As described by Potocnjak, P., et al., (1980) J. Exp. Med. 151:1504-13, and Yoshida, N., et al., (1980) Science 207:71-73, for the spozozoite of malaria, monoclonal antibodies against different antigens can be screened to determine which one confers protection, and that specific monoclonal antibodies can be used to purify the surface antigen and produce a vaccine. Monoclonal antibodies have been generateo against a number of parasites whose biochemistry and mechanism of pathogenesis can now be more quickly explored. Pearson, T. et al., (1980) J. Immunol. Methods 34:141-54; Sethi, K. et al., (1980) J. Parasitol. 66:192-96.
In most cases, the hybridomas producing these monoclonal antibodies have been obtained by the fusion of mouse myelomas and antigen-stimulated mouse lymphocytes isolated from the spleen. While useful to a certain extent in antigenic analyses, monoclonal antibodies of rodent origin directed against human parasites suffer at least two shortcomings. First, rodents do not necessarily respond to the same antigenic determinants of the parasite as do humans. See Sikora, K. and Wright, R. (1981) Br. J. Cancer 43:696-700. Second, rodent monoclonal antibodies would have limited use in human immunotherapy because they are viewed by the human immune system as foreign, and are rejected. Olsson, L. and Kaplan, H. (1980) Proc. Natl. Acad. Sci. USA 77:5429-5431.
Attempts at overcoming the above-described shortcomings have focused on hybridizing antigen stimulated human lymphocytes with either human or mouse myelomas. The production of human-human intraspecies hybridomas has been hampered mainly by the current scarcity of human myeloma cell lines, which when fused, will support the production of immunoglobulin. Kozbor, D. and Roder, J. (1983) Immunol. Today 4:72-79. In addition, there are constraints on how the lymphocytes of humans can be ethically stimulated and obtained.
To overcome the lack of human-derived cell fusion partners, human lymphocytes have been fused with mouse myeloma cell lines to yield mouse-human interspecies hybrids. Such hybridomas have been made to secrete human antibody against the Forssman antigen [Nowinski, R. et al., (1980) Science 210:237-239], human mammary carcinoma cells [Schlom, T. et al., (1980) Proc. Natl. Acad. Sci. USA 77:6841-6845], keyhole limpet hemocyanin [Lane, H. et al., (1982) J. Exp. Med. 155:333-338] and tetanus toxoid [Kozbor, D. et al., (1982) Hybridoma 1:323-328].
However, it has been found that most mouse-human interspecies hybridomas preferentially segregate human chromosomes, thereby making preparation of stable lines secreting human antibody a labordous task. Such loss of human chromosomes from mouse-human hybridomas is not random. It is known that human chromosomes 14 (heavy chain) and 22 (light chain-lambda) are preferentially retained whereas chromosome 2 (light chain-kappa) is preferentially lost.
Even hybrids possessing the appropriate human chromosomes often fail to secrete human immunoglobulin because the appropriate environmental stimuli are absent. Kozbor, D. and Roder, J., supra. In addition, mouse-human hybrids that do produce antibodies are reported to secrete mouse immunoglobulins and/or immunoglobulins that are part mouse and part human. Schwaber, J. (1975) Exp. Cell Res. 93:343-354.
U.S. Pat. Nos., 4,172,124 and 4,196,265 to Koprowski et al. disclose the production of monoclonal antibodies that immunoreact with tumors and viruses, respectively. Both patents teach that the species from which the antibody-producing cell and myeloma cell are derived are unimportant to the production of the resulting, fused hybridoma cells. Those teachings nothwithstanding, those patents only specifically disclose the preparation of mouse-mouse hybridomas, and those skilled in the art are aware that the species of the lymphocyte and myeloma cell lines are important to the successful production of useful hybridomas and of their monoclonal antibodies.
The present invention, described hereinafter, resulted from study of parasitic microorganisms of the genus Plasmodium. The genus is currently defined on the basis of one type of asexual multiplication by division occurring in the parenchymal cells of the liver of the vertebrate host (exo-erytahrocytic schizogony); the other characteristic is that the mosquito host is a species of Anopheles. L. J. Bruce-Chwatt. Essential Malariology, William Heinemann Medical Books, Ltd., London (1980) Chapter 2. The four generally recognized species of Plasmodia infecting man are P. malariae, P. vivax, P. falciparum and P. ovale.
The life cycle of all species of human malaria parasites is essentially the same. It comprises an exogenous sexual phase (sporogony) with multiplication in certain Anopheles mosquitos, and an endogenous asexual phase (schizogony) with multiplication in the vertibrate host. The latter phase includes a developmental cycle in the red corpuscles in the blood (erythrocytic schizogony) as well as the cycle taking place in the parenchymal cells of the liver (exo-erythrocytic schizogony).
In the early phases of erythrocytic schizogony, the parasites are termed trophozoites. After a period of growth the trophozoites multiply by the asexual dividing process of schizogony. Mature schizonts are fully developed forms in which, as a result of the segmentation of the nucleus and the cytoplasm, a number of small rounded forms termed merozoites are produced.
When the process of schizogony is completed, the red blood cell bursts and the merozoites then invade fresh erythrocytes in which another generation of parasites is produced by the same process. This process is repeated over and over again in the course of infection leading to a progressive increase of parasitemia until the process is slowed down by the host's immune response.
Of all the species of human Plasmodia, P. falciparum is the most highly pathogenic. A P. falciparum infection in non-immune subjects usually runs an acute course, and frequently terminates fatally unless promptly treated with specific drugs.
Currently, the only certain means of diagnosing malarial infection is the detection of the Plasmodium by microscopical examination of the blood. The thick film method is recommended because it concentrates by a factor of 20-40 the layers of red blood cells on the microscope slide surface, and thereby reveals even scanty infections within a short time. While the parasites are easily detected in the thick film assay, they are very difficult to identify as to species using this method.
Since species identification may be clinically important, the thick film assay must be supplemented by a thin film assay that allows for species identification. Standard practice requires that an experienced technician examine an appropriately stained thick film for at least 5 minutes (corresponding to approximately 100 microscopic fields under oil immersion); thin films must be examined for 15-20 minutes before a negative report is justified. In doubtful cases repeated blood films must be taken and examined every 4 hours, resulting in a significant investment of technician time. The thick and thin film methods are described by L. J. Bruce-Chwalt in Essential Malariology, William Heinemann Medical Books, Ltd., London (1980) pages 76-96.
Assays using immunofluorescence, immuno-haemagglutination, immuno-precipitation and enzyme-linked immunosorbent methods have been used widely for the detection and measurement of anti-malarial antibodies. However, these serological tests are of limited use for the diagnosis of acute malaria since they become positive only several days after the appearance of malarial parasites in the blood.
Once malaria has been diagnosed there exists a spectrum of antimalarial drugs that may be used to intervene on different phases of the parasite life cycle. The state of the infected person's immunity has a bearing on the use of drugs since persons who have acquired a degree of immunity through exposure can be cured or protected from serious symptoms more easily than those who have not. However, to date there is no drug available that provides absolute protection from initial infection.
Despite the remarkable progress in the study of immune responses in malaria there still exists no vaccine against the parasite. Natural immunity in malaria is directed against the asexual erythrocyte forms of the parasite (i.e., trophozoite, schizont ano merozoite), but not necessarily against sporozoites. However, further progress in this area has been hampered by the inability to produce antisera specific for host-recognized antigenic determinants unique to these life cycle stages.